African Journal of Biotechnology Vol. 10(55), pp. 11345-11359, 21 September, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.1416 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Bioinformatic identification of microRNAs and their targets in Aquilegia formosa x Aquilegia pubescens Yongzhong Lu* and Zhijian Zhang Biology Department, Qingdao University of Science and Technology, Qingdao 266042, China. Accepted 29 November, 2010 As a model system, Aquilegia is of evolutionary and ecological significance. Availability of new genomic resources is facilitating the related researches at molecular level. MicroRNAs (miRNAs) are a class of endogenous, non-coding and short RNAs directly involved in regulating gene expression at the post-transcriptional level. High conservation of miRNAs in plants provides the foundation for identification of conserved miRNAs in other plant species through homology alignment. For the purpose of finding miRNAs in Aquilegia Formosa x Aquilegia pubescens, previous known plant miRNAs were, plant were used for BLAST search against its expressed sequence tag (EST) database and following a series of filtering criteria, 12 new miRNAs belonging to 5 miRNA families were identified while 51 potential target genes were subsequently predicted, most of which seemed to encode transcription factors or enzymes participating in regulation of development, growth, metabolism and other physiological processes. These findings not only lay the foundation for understanding the roles of miRNAs in Aquilegia, but also provide a phylogenetically important dataset for plant miRNA evolution studies. Key words: Aquilegia, bioinformatic analysis, microRNA, evolution. INTRODUCTION The lower eudicot genus Aquilegia represents a phylo- hybridization in adaptive radiation and evolution has been genetic midpoint between the eudicot and monocot debated for decades, recent molecular genetic studies models such as Arabidopsis and Oryza, and holds enor- have indicated that hybridization is surprisingly frequent mous potential for investigating aspects of development, in natural populations, which can lead to rapid genomic ecology and evolution (Kramer, 2009). Besides, species changes, including chromosomal rearrangements, geno- in this flowering plant genus have undergone a very re- me expansion, different gene expression and silencing cent adaptive radiation and present a unique opportunity and the beneficial new phenotypes (Rieseberg, 2009). to investigate the molecular genetic changes underlying Availability of genomic data will produce a new under- adaptations (Kramer, 2009; Puzey and Kramer, 2009). standing of the genetic nature of species and will help Aquilegia formosa and Aquilegia pubescens are two resolve a century-old debate over the role of hybridization closely related species belonging to the columbine genus (Baack and Rieseberg, 2007). (Cooper et al., 2010). Despite their morphological and MicroRNAs (miRNAs) are a class of endogenous, ecological differences, hybrid population can form when small, non-coding and single-stranded RNAs that act as hybrid zone is established. Though, the importance of post-transcriptional regulators in eukaryotes (Unver et al., 2009). They can control many important aspects of plant development, suggesting that these molecules may also have played key roles in the evolution of developmental *Corresponding author. E-mail: [email protected]. Tel: +86-532- processes in plants (Jasinski et al., 2010). In recent 84022845. years, the evolution and conservation of plant miRNAs has been the subject of significant investigation (Axtell Abbreviations: miRNAs, MicroRNAs; ESTs, expressed and Bowman, 2008). Although the roles of miRNAs have sequence tags. been extensively studied, their expression diversity and 11346 Afr. J. Biotechnol. evolution in closely related species and interspecific Local Alignment Search Tool (BLAST) search against the EST hybrids are poorly understood (Ha et al., 2009). database, with the BLASTN parameters Evalue being 1000 and In Aquilegia, miRNAs only have been reported in word-match size between the query and database sequences being 7. Mature miRNA sequences should be no less than 16 nt and the Aquilegia coerulea (Puzey and Kramer, 2009). Availability mismatches should be less than 4. Wherever available, precursor of new genetic and genomic resources, especially the sequences of 400 nt were extracted (200 nt upstream and 200 nt publishing of EST database of A. formosa x A. pubescens downstream to the BLAST hits) and used for the hairpin structure (http://www.ncbi.nlm.nih.gov/) provides the chance to prediction. If the length of a sequence was less than 400 nt, the investigate the expression diversity and evolution in these entire available sequence was used as a miRNA precursor sequence. These precursor sequences were screened by BLASTx closely related species and interspecific hybrids. Nowa- online to reject the protein coding sequences days, two major categories of approaches have been (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The retained precursor applied for miRNAs investigation (Unver et al., 2009). sequences underwent hairpin structure prediction through web Compared to the experimental approaches, computa- server mfold. Only those meeting the following criteria were tional methods have been proved to be faster, more designated as miRNA homologs: (1) RNA sequence folding into an affordable and more effective, contributing mostly to appropriate stem-loop hairpin secondary structure; (2) a mature miRNA sequence located in one arm of the hairpin structure; (3) today’s plentiful storage in miRBase (Unver et al., 2009). predicted mature miRNAs with no more than 3 nt substitutions as Different computational miRNA finding strategies have compared with the known miRNAs; (4) miRNAs having less than 6 been developed based on a core principle of looking for mismatches with the opposite miRNA* sequence in the other conserved sequences among different species that can strand; (5) no loop or break in miRNA* sequences; (6) predicted fold into extended hairpins (Bonnet et al., 2004). The secondary structures having higher minimal folding free energy biogenesis of miRNAs suggests that it is possible to find index (MFEI)(absolute value), which usually being over 0.85 (Zhang et al., 2006b). Also, the AU content of pre-miRNA should be miRNAs by searching expressed sequence tags (ESTs) between 30 and 70% (Xie et al., 2007). with known miRNAs. There have been more and more reports about the identification of miRNAs by mining the repository of available ESTs (Lu and Liu, 2010a; Han et Prediction of miRNA targets al., 2009; Zhang et al., 2009; Song et al., 2009; He et al., The near-perfect complementarity of plant miRNAs for their targets 2008; Xie et al., 2007; Zhang et al., 2008). EST analysis allows for very accurate prediction of miRNA targets (Fahlgren et makes it possible to rapidly study miRNAs and their al., 2010). MiRNA targets prediction was performed by aligning the functions in species whose genome sequences have not predicted miRNA sequences with EST sequences of A. formosa x been well known (Zhang et al., 2006a). A. pubescens via the BLASTN program. The targets were screened The goal of this study is to identify new miRNAs from according to these criteria: the number of mis-matches should be the EST sequences of A. formosa x A. pubescens throu- less than 4 and no gaps were allowed at the binding site. The predicted target ESTs for each miRNA family were also aligned gh bioinformatic analysis. The findings will lay foundation against one another in order to eliminate redundancies (ESTs that for further research of the roles of miRNAs in Aquilegia shared greater than 98% sequence identity, usually due to separate and also will provide a phylogenetically important dataset annotations of alternative splicing products of the same locus). After for plant microRNA evolution studies. removal of the repeated sequences, the function of the potential target genes were predicted by BLASTX against non-redundant protein sequences database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Identity > 25%). MATERIALS AND METHODS Sequences and softwares Phylogenetic analysis of the new miRNAS The known plant miRNA sequences from Arabidopsis, Brassica, Considering the conservation of miRNAs and their precursors, the Glycine, Saccharum, Sorghum, Vitis, Solanum, Oryza, Triticum, precursor sequences of the novel and the known miRNAs in the Chlamydomonas and other plant species were downloaded from same family were aligned and phylogenetically analyzed by the miRNA database miRBase (http://www.mirbase.org) (Release ClustalW online to investigate their evolutionary relationships 14: September 2009). After removal of the repeated sequences, (http://www.clustal.org/). 2177 items were left as the reference set. The 85041 EST sequences of A. formosa x A. pubescens and 12313 GSS sequences of A. formosa and Aquilegia vulgaris were downloaded RESULTS AND DISCUSSION from GenBank (http://www.ncbi.nlm.nih.gov/), Blast-2.2.21-ia3 was downloaded from NCBI (http://www.ncbi.nlm.nih.gov/Ftp/) and set up locally. RNA secondary structure and the free energy were Identification of A. formosa x A. pubescens miRNAs calculated by web server mfold (http://mfold.bioinfo.rpi.edu/) (Zuker, 2003). The software MiRNAassist was applied to improve the Sequence and structure homologies are the main theory analysis efficiency (Xie et al., 2007). behind the computer-based approach for miRNAs prediction.